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Potassium movements in denervated frog sartorius muscle.

The movement of 42K+ across the sarcolemma and the resting membrane potential (VM) of normal and denervated frog sartorius muscle were studied under several experimental conditions in preparations initially equilibrated in 100 mM K+ and 219 mM Cl-. The results can be summarized as follows. In the absence of any driving force on K+, i.e., when the difference between VM and the K+ equilibrium potential (EK) is zero (VM - EK = 0), the K+ conductance (gK) was 368 microseconds . cm-2 in control and 282 microseconds. in denervated muscle. The reduced gK of denervated muscles results from the addition of the opposite changes in the conductances of a Rb+-sensitive inward rectifying pathway (gIR), which decreases, and a Rb+-insensitive linear channel (gL), which increases. Thus in control muscles gK (368 microseconds . cm-2) equals gIR (359 microseconds . cm-2) plus gL (9 microseconds . cm-2), while in denervated muscles gK (282 microseconds . cm-2) equals gIR (198 microsecond . cm-2) plus gL (84 microseconds . cm-2). Denervation significantly reduces the inward rectifying properties of the resting K+ permeability system. In the presence of outward driving forces on K+ (VM - EK greater than 0) of 35-50 mV, the Rb+-sensitive inward rectifier channel appears to close completely in both control and denervated muscles. In the latter, however, the effect was not as well maintained as in the former, suggesting that its closing mechanism might be altered by denervation. No changes were observed during the first 2 wk after denervation.

Animals↗

Radioactive calcium influx at rest and during potassium contracture in the T-disrupted, and the urea- or manganese-treated frog sartorius muscles.

45Ca influx in the intact, the T-disrupted, and the urea-treated or Mn++-treated frog sartorius muscles was measured at rest and during potassium contracture. In the intact muscle, Ca influx increased with the increasing of potassium concentration in the bathing solution, and the time course of the Ca influx showed considerable correspondence with that of the potassium contracture. This increased Ca influx was divided into two components: early rapid Ca influx and late slow Ca influx. The amount of early rapid Ca influx at higher concentrations of potassium occupied about 60% of the total Ca influx. The early rapid Ca influx markedly decreased in the T-disrupted and in the urea-treated or Mn++-treated muscles. On the other hand, the late slow Ca influx and the resting Ca influx in these muscles changed only slightly. These results suggest that the early rapid Ca influx occurs mainly through the membrane of the T-system, particularly at the triadic junctional area of muscle fiber, while the late slow Ca influx and the resting Ca influx occur mainly through the surface membrane.

Animals↗

An analysis of the action of atropine and scopolamine on the end-plate current of frog sartorius muscle.

The effects of atropine sulfate and scopolamine hydrobromide were investigated on the end-plate current (EPC) of frog sartorius muscle by standard voltage-clamp techniques. Both atropine and scopolamine reduced the peak EPC amplitude although scopolamine was only one-third as potent as atropine. The reduction of amplitude became more pronounced with increasing membrane hyperpolarization resulting in nonlinear current-voltage characteristics. Atropine shortened the EPC duration and decreased the voltage-sensitivity of the falling phase; the latter, however, continued to remain a single exponential function of time as in the control. Scopolamine reduced the time to peak, and in addition, converted the falling phase to a double exponential function, consisting of a rapid initial phase followed by a slow terminal phase. Both phases of fall were altered by changes in drug concentration but only the terminal phase responded appreciably to changes in membrane potential. Atropine and scopolamine were without effect on the EPC reversal potential, indicating that the drugs do not exhibit a preference for the ionic species carrying the synaptic current.

Animals↗

[Analysis of frequency facilitation in neuromuscular synapses of the sartorius muscle in frogs].

Changes in the quantum content of end plate potentials (EPPs) were studied in experiments on the frog sartorius muscle in the course of a gradual increase in stimulation frequency (frequency facilitation). It was shown that the frequency facilitation coefficient does not depend on the initial quantum content of EPPs but is in good agreement with the initial frequency of miniature EPPs and in bad agreement with their amplitude. The frequency facilitation is accompanied by the growth of the statistical binomial parameters P and n. The increase in P parameters is consequent of interference in P changes during the second component of facilitation after the single impulse. The growth of n is accounted for by the summation of the n increase during the first component of facilitation the time course of which is prolonged on repetitive stimulation.

Animals↗

High resolution scanning electron microscopy of frog sartorius muscle.

A field emission-type scanning electron microscope (SEM) was used to study the three-dimensional ultrastructure of frog sartorius muscles. Various preparative procedures were tested to seeks better specimen preparation for high resolution SEM observation. Procedures should be chosen depending on the information desired. The cell surface and intracellular organization of muscle fibers were best visualized when the tissues were fixed with tannic acid-OsO4 and torn after critical point drying. The basal lamina appeared as a continuous felt-like layer, onto which fine filamentous materials adhered. The true outer surface of the sarcolemma was not seen, whereas the true inner surface was occasionally exposed and exhibited numerous caveolae, membraneous fragments and fine filaments attached to its surface. In freeze-fractured and dried tissues, the cleaved sarcolemma showed numerous apertures of caveolae and T-system tubules. Inside the cell, the myofibrils showed a typical branding pattern of the sarcomere. Thick myofilaments were regularly beaded except for the pseudo-H-zone. Around the myofibrils the sarcoplasmic reticulum and T-system were also clearly observed. The results are discussed with special reference to the usefulness and limitation of the high resolution SEM in studying the ultrastructure of cells and tissues.

Animals↗

Effect of prostaglandins E1 and F2 alpha on neuromuscular transmission in the frog sartorius muscle.

End plate potentials (e.p.p.s.) and miniature end plate potentials (m.e.p.p.s.) were recorded intracellularly at the neuromuscular junction of the frog sartorius muscle. Addition of as little as 8.5 x10(-8)M PGE1 reduced the mean m.e.p.p. frequency. The mean amplitude of m.e.p.p.s was not changed, the mean amplitude of the e.p.p.s and the quantum content of the transmitter released by a nerve impulse was slightly reduced. A decrease in mean m.e.p.p. frequency was also seen in response to the administration of 8.5 x 10(-8)M PG2 alpha. The mean amplitude of e.p.p.s and m.e.p.p.s and the quantum content remained unchanged. The possible presynaptic mode of action of PGs in the preparation of discussed.

Animals↗

Inhibition of sodium for sodium exchange by phlorizin in frog sartorius muscle.

The effects of phlorizin (2 X 10(-3) mol X l-1) on the Na transport of frog (Rana esculenta) sartorius muscle were investigated in glucose-free medium. Phlorizin decreased the rate coefficient of 24Na efflux by about 40%. The degree of inhibition was comparable to that caused by ouabain (10(-4) mol X l-1). Phlorizin could evoke a further reduction in the 24Na efflux also in the presence of ouabain. The intracellular Na content of the phlorizin-treated muscles remained unchanged, in contrast to a 60% increase induced by ouabain. 42K uptake was not affected by phlorizin. Data indicate that the ouabain-sensitive Na-K pump was not involved in the action of phlorizin. At the same time, phlorizin failed to alter the residual 24Na efflux measured in Li-Ringer solution containing ouabain. When Na: Na exchange was restored by replacing Na into the washout solution in the presence of ouabain, the increase of 24Na efflux was significantly diminished by phlorizin. Phlorizin reduced the 24Na uptake into a compartment with a half time of 6 min by about 40% without affecting the intracellular compartment. The results suggest that phlorizin inhibits the ouabain-insensitive Na: Na exchange in a superficial Na compartment.

Animals↗

Action of choline on frog sartorius muscle.

1. External and micro-electrode measurements were made of fibre membrane potentials taken at intervals along the length of frog sartorius muscle fibres before and after adding choline. In ;cold-treated' Rana pipiens which had been kept at 1-3 degrees C for at least 5 days, 10(-3) w/v choline caused a long-lasting depolarization of approximately 3 hr. Depolarization occurred first at the end-plate region and spread more slowly to the end-plate free region. The potential became equalized along the muscles during recovery.2. R. pipiens kept at room temperature showed a faster repolarization in the choline solution, within approximately 1 hr. R. temporaria showed a similar recovery time in muscles from ;cold-treated' animals. In R. temporaria kept at room temperature the duration was shorter, approximately 20-30 min. The apparently generalized depolarization of the muscle seen in ;cold-treated' R. pipiens appears to be due to the spreading effect of a long-maintained depolarization at the end-plates. A similar far-spread of depolarization was seen following crush of fibres.3. Length-constants of muscle fibres determined with pulses were found to be within the normal range in muscles from ;cold-treated' R. pipiens and slightly reduced after adding choline.

Animals↗

Lifetime and conductance of acetylcholine-activated channels in normal and denervated toad sartorius muscle.

1. The average lifetime and conductance of acetylcholine-activated channels were measured in normal and denervated, voltage-clamped toad sartorius muscle fibres at 10 degrees C. 2. The null potential was -4 +/- 1 mV for subsynaptic channels in normal fibres and -6 +/- 3 mV for extrasynaptic channels in denervated fibres. 3. There was a linear relationship between variance of conductance fluctuations and mean conductance for acetylcholine-induced currents up to 50 nA, in denervated fibres clamped at -50 mV. The ratio gave a channel conductance of 14 pS. 4. At the same membrane potential, the average lifetime of extrasynaptic channels in denervated fibres was approximately double, whereas channel conductance was approximately half, that of subsynaptic channels in normal fibres: there was little difference in net charge transfer through the two types of channel under similar conditions. 5. Single channel conductance increased, whereas average channel lifetime decreased, as the membrane potential became more positive (depolarized). The effect of potential on channel lifetime and conductance was more pronounced in denervated than in normal fibres.

Acetylcholine↗

Tolbutamide, but not glyburide, affects the excitability and contractility of unfatigued frog sartorius muscle.

The goal of this study was to characterize the effects of tolbutamide and glyburide, two known KATP channel blockers, on intact, unfatigued sartorius muscle fibres of the frog, Rana pipiens. Tetanic contractions were elicited by field stimulation with 200 ms long train of pulses (0.5 ms, 6 V, 140 Hz). Resting and action potentials were measured using conventional microelectrodes. At pHo 7.2 (extracellular pH), the tetanic force was unaffected by 0.5 mM and 1.0 mM tolbutamide, but at 2.0 mM it decreased by 15.5 +/- 1.0%. The effect of tolbutamide on the tetanic force was significantly greater at pHo 6.4: all three tolbutamide concentrations caused a significant decrease in tetanic force, being 62.3 +/- 9.4% at 2 mM. In the presence of tolbutamide a large number of fibres became unexcitable at pHo 6.4, but not at pHo 7.2. Glyburide at 10 microM, on the other hand, caused a 5-7% decrease in tetanic force at both pHo 6.4 and 7.2, but no further decreases in tetanic force were observed when the glyburide concentration was increased up to 100 microM. Unlike tolbutamide, glyburide did not affect the excitability of muscle fibres, but significantly prolonged the repolarization phase of action potentials, especially at pHo 6.4. We suggest that several of the tolbutamide effects reported in this study cannot be accounted for by a direct effect on KATP channels, and that the large decrease in membrane excitability and muscle contractility in the presence of tolbutamide must seriously be taken into consideration when this channel blocker is used to study the physiological role of KATP channels in intact muscle fibres.

Action Potentials↗

Kinetics of oxygen consumption after a single isometric tetanus of frog sartorius muscle at 20 degrees C.

The time-course of the rate of oxygen consumption (QO2) has been measured in the excised frog sartorius muscle after single isometric tetani of 0.1-1.0 s at 20 degrees C. To measure deltaQO2(t), the change in QO2 from its basal level, a novel method was devised, based on the validity in this tissue of the one-dimensional diffusion equation for oxygen, established in the preceding paper. After a tetanus, deltaQO2 reached a peak within 45-90 s, then declined exponentially, and could be well fit by deltaQO2(t) = QO + Q1(epsilon -k1t - epsilon-k2t). tau2 (= 1/k2), which characterized the rise of deltaQO2, was a decreasing function of tetanus duration (range: from 1.1 +/- 0.28 min [nu = 5] for a 0.1-s tetanus, to 0.34 +/- 0.05 min [nu = 8] for a 1.0-sec tetanus). tau1 (= 1/k1), which characterized the decline of deltaQO2, was not dependent on tetanus duration, with mean 3.68 +/- -.24 min (nu = 46). A forthcoming paper in this series shows that these kinetics of deltaQO2 are the responses to impulse-like changes in the rate of ATP hydrolysis. The variation of tau2 with tetanus duration thus indicates the involvement of a nonlinear process in the coupling of O2 consumption to ATP hydrolysis. However, the monoexponential decline of deltaQO2(t), with time constant independent of tetanus duration, suggests that during this phase, the coupling is rate-limited by a single reaction with apparent first order kinetics.

Animals↗

The relations between sarcomere length and characteristics of isometric twitch contractions of frog sartorius muscle.

1. Relations between sarcomere length, tension and time course of isometric twitches at 20 degrees C were determined for thirty-two sartorius muscles from Rana temporaria.2. The maximum isometric twitch tension per unit cross-sectional area of muscle ranged from 0.56 to 2.2 kg/cm(2) at 20 degrees C and initial sarcomere length about 2.1 mu. This variation was not correlated with the corresponding measure of tetanic tension.3. The maximum isometric twitch tension per unit cross-sectional area of muscle at 2.1 mu sarcomere length was directly correlated with twitch contraction time and inversely correlated with the cross-sectional area of the muscle.4. The isometric twitch contraction was potentiated with increase in initial sarcomere length from about 2.1 to about 2.8 mu at 20 degrees C, and the degree of potentiation was inversely correlated with the maximum isometric twitch tension per unit cross-sectional area of muscle at 2.1 mu sarcomere length.5. The sarcomere length: twitch tension relation is labile and may be altered by changes in temperature and the after-effects of repetitive stimulation.6. Variation in twitch contractions at different sarcomere lengths are discussed in connexion with excitation-contraction coupling and the degree of activation of muscle fibres during twitch contractions at 20 degrees C.

Animals↗

The pattern of activation in the sartorius muscle of the frog.

The development of isometric twitch tension has been compared with the redevelopment of isometric tension in the fully active frog sartorius muscle following release. At 0 degrees C the rate of rise of isometric twitch tension is the same as that for the muscle in the fully active state at the same tension but not until about 40 msec. after the stimulus and then only for a few milliseconds. The rates of rise of tension in the twitch and in the redevelopment of tension in the fully active muscle appear to be nearly the same at low tensions. Substitution of nitrate for chloride in the Ringer's solution bathing the muscle retards the development of tension during the early part of the contraction phase of the twitch and the effect reaches a maximum within 3 minutes after changing the solutions. These observations have been discussed in connection with some possible patterns of activation and the hypothesis has been advanced that the rate of activation of a sarcomere is determined mainly by the rate at which the transverse component of the link between excitation and contraction is propagated inwards from the periphery to the center of the fiber. This hypothesis has been discussed in relation to others concerning the nature of excitation-contraction coupling.

Animals↗

Effect of oxygen on blood flow autoregulation in cat sartorius muscle.

To assess the role of O2 in blood flow autoregulation, arteriolar diameter and erythrocyte velocity were measured in individual microvessels of the cat sartorius muscle while ambient O2 tension (PO2) and perfusion pressure were altered. The muscle surface was covered with a layer of silicone fluid equilibrated with a gas mixture containing 0--20% O2. Under control conditions (0% O2) all except the largest arterioles dilated with pressure reduction, and all showed significant blood flow autoregulation. Elevated PO2 diminished flow regulation and dilation in large and small arterioles when arterial pressure was reduced. This effect was generally more pronounced in the small arterioles where elevated PO2 caused complete cessation of blood flow. Complete blood flow stoppage was not routinely seen in larger vessels and may reflect the fact that these vessels also supply deeper tissue regions less affected by the change in ambient PO2. Our results indicate that the PO2 level of the tissue may be an important determinant in blood flow autoregulation.

Adaptation, Physiological↗

[Effect of amines on insulin-stimulated processes: the transport of sugars and synthesis of glycogen in frog sartorius muscles].

Amines and amides were found to inhibit the process of stimulation of sugar transport in muscle tissue (N. A. Vinogradova et al., 1978). The present paper reports results of experiments on frog sartorius muscles acted upon by amines and other substances that inhibit induction of cultured cell proliferation. The stimulation of sugar transport induced by insulin, 2,4-dinitrophenol, or KCl was found to be inhibited by dansylcadaverine (1 mM), 5-methoxytryptamine (2 mM), or methylamine (100 mM). Such substances as chloroquine, bacitracin, or monensin exerted no effect. Besides, dansylcadaverine (1 mM), and 5-methoxytryptamine inhibited the stimulation of insulin induced glycogen synthesis. Dansylcadaverine was ineffective at concentrations lower than 0.5 mM. It is suggested that the inhibitory action of amines depends on their influence on the processes of membrane protein phosphorylation.

Amines↗

Fractionation of sodium effux in frog sartorius muscles by strophanthidin and removal of external sodium.

The influence of strophanthidin, ouabain, and the removal of external sodium on the sodium efflux from frog sartorius muscle was measured. In freshly dissected muscles strophanthidin and ouabain in maximally effective concentrations reduced the efflux of sodium by about 50%. Of the sodium efflux which is strophanthidin-insensitive about 75% is inhibited after complete replacement of external sodium by lithium. In the absence of strophanthidin replacement of external sodium by lithium, calcium, or magnesium produces an initial rise in the sodium efflux, followed by a fall in the efflux as the exposure of the muscles to sodium-free media is continued. When the muscles are exposed for prolonged periods in sodium-free media, the fraction of internal sodium lost per minute is higher when returned to normal Ringer fluid than it was initially. The activation of sodium efflux by external sodium after long periods in sodium-free solutions is partly strophanthidin-sensitive and partly strophanthidin-insensitive. The internal sodium concentration is an important factor in these effects. The effects of temperature on the sodium efflux were also measured. Above 7 degrees C the Q(10) of both the strophanthidin-sensitive and strophanthidin-insensitive sodium efflux is about 2.0. Below 7 degrees C the strophanthidin-insensitive sodium efflux has a Q(10) of about 7.4.

Animals↗